Rapid Enantiomeric Ratio Determination of Multiple Amino Acids Using Ion Mobility-Mass Spectrometry

Chiral analysis is becoming increasingly important across various scientific fields, including chemistry, pharmaceuticals, biosciences, and more recently, metabolomics. In this context, a high-resolution and high-throughput method was developed for the simultaneous determination of the enantiomeric ratio (<i>er</i>) of seven pairs of amino acid (AA) enantiomers (Arg, Gln, His, Met, Pro, Tyr, and Trp) using flow injection analysis coupled with ion mobility-mass spectrometry (FIA-IM-MS) technology. Specifically, the Single Ion Mobility Monitoring (SIM²) mode on a TIMS-Tof^TM instrument enabled the rapid relative quantification of chiral compound mixtures. A linear model accurately described the relationship between enantiomeric ratio and IM-MS response for Arg, Gln, and Pro enantiomers, as evidenced by high R² values and unbiased residuals. In contrast, non-linear trends were observed for His, Tyr, and Trp, where a quadratic model significantly improved the fit. However, the linear model was retained for Met, despite an R² of about 0.98, due to its comparable performance and simplicity. Measurement accuracy was confirmed with very good recovery rates for <i>er</i> values of 0.95 and 0.99 across all AAs. Finally, the potential of the FIA-SIM²-MS approach in chiral analysis was demonstrated, particularly its ability to provide a reliable and efficient high-throughput tool for accurate <i>er</i> determination.